Measurement of pO2 in cultured mouse oocytes using electron paramagnetic resonance oximetry

Abstract

We investigated the feasibility of measuring the partial pressure of oxygen (pO 2 ) in cultured mouse oocytes by electron paramagnetic resonance (EPR) oximetry. Approximately 15 pL (0.015 μL) of Ox063, an oxygen-sensing paramagnetic material, at a concentration of 500 mM was injected into the ooplasm of mouse oocytes. When one, five and 20 oocytes were used, the sample of 20 oocytes was sufficient to yield an effective EPR spectrum for determining the pO 2 . The mean pO 2 levels in oocytes cultured under tensions of 5 and 20% O 2 were 60.2 and 153.1 mmHg, respectively. The present study indicates that it is possible to utilize EPR oximetry with Ox063 for the measurement of pO 2 in cultured oocytes.Oxygen tensions in follicular fluid (4) and the female reproductive tract (3) are ≤ 40% of atmospheric oxygen. Therefore culture and handling of oocytes/ embryos under the atmospheric level of oxygen (about 21% O 2 ) results in oxidative stress to them through the excessive generation of reactive oxygen species (ROS) (1, 5). Reduced oxygen tension (5% O 2 ) in the gas phase for assisted reproductive technology (ART) such as in vitro fertilization (2) and in vitro embryo culture (6) was reported to be beneficial to embryo development. However, we need to handle and manipulate oocytes/embryos repeatedly in air during ART procedures such as transferring oocytes/embryos between culture dishes and intracytoplasmic sperm injection. These procedures may increase the partial pressure of oxygen (pO 2 ) and the level of ROS in oocytes/embryos. Therefore a method for repetitive measurement of the pO 2 in oocytes/ embryos during ART procedures could be useful not only for monitoring the culture and handling conditions but also for improving the outcome of ART through examining the relationship between pO 2 and the level of oxidative stress in oocytes/embryos. To our knowledge, data on pO 2 of mammalian oocytes/embryos are limited. In the hamster, pO 2 levels in oocytes and 2-to 8-cell stage embryos recovered from the oviducts were measured using a microelectrode technique (12). However, there is no information on the pO 2 in mammalian oocytes/embryos cultured or manipulated in vitro. For measuring the pO 2 in tissues, several methods have been developed but most techniques are limited in their ability to analyze a single cell such as an oocyte (13). Among the several known oximetry techniques, a method based on electron paramagnetic resonance (EPR) called EPR oximetry is minimally invasive, accurate, and allows for repeated measurements, and so has been utilized in many tissues (7) and cell suspensions (11). EPR oximetry uses exogenous oxygen-sensing paramagnetic probes whose EPR line widths are broadened linearly with respect to the pO 2 levels in the vicinity. Thus, the measured spectral line width can be converted to the oxygen concentration using an appropriate standard curve. Among oxygen-sensing probes, the triarylmethyl radical Ox063 is a biologically stable and nontoxic molecule, and cannot permeate the cell membrane

Microarray analysis of mRNA from cumulus cells following in vivo or in vitro maturation of mouse cumulus–oocyte complexes

“…This leads to an interesting conundrum: COCs within antral follicles should theoretically experience hypoxic conditions, but appear not to. Interestingly, using electron paramagnetic resonance oximetry with a unique oxygen probe injected within denuded oocytes, Higaki et al (2010) recently revealed that oocytes incubated under 35 mmHg O 2 (5%) had an intracellular O 2 concentration of 50 mmHg, supporting the notion that O 2 sequestering may indeed occur within oocytes and cumulus cells.…”

Complexes of IrIII‐Octaethylporphyrin with Peptides as Probes for Sensing Cellular O2

“…9. In recent years, new methods for minimally invasive sensing of intracellular O 2 (icO 2 ) were introduced which use nitroxyl and esterified trityl (triarylmethyl) radicals,5, 6, 10, 11 O 2 -sensitive genetically encoded GFP constructs,12 endogenous mitochondrial protoporphyrin IX,13, 14 Ir-BTP, Ru–polypirydyl, or Pt– and Pd–porphyrin probes based on cell-penetrating peptide(s) or nanoparticles 15–27…”